The long-term goal of our research is to provide the mechanistic basis for the development of an effective immunotherapy for cancer using the natural and engineered properties of V?24-invariant Natural Killer T cells (iNKTs). In the course of the mechanistic studies supported by this grant we have found that iNKTs traffic toward NB in response to tumor-derived chemokines and mediate anti-tumor activity indirectly via targeting of tumor-associated macrophages (TAMs). We also demonstrated that TAMs infiltrate primary tumors in a subset of NB patients and the presence of M2-like TAMs is associated with a novel inflammatory signature that serves as an independent prognostic factor of poor outcome. However, the mechanisms responsible for the initiation and regulation of tumor-supporting inflammation in NB are unknown and will be addressed in the renewal application. In the search for the molecular triggers of an inflammatory response in NB, we found that a subset of NB cells in all examined human and murine cell lines, as well as primary human and transgenic murine tumors, expresses a transmembrane form of TNF? (tmTNF?). Recent reports suggest that unlike soluble (s)TNF?, tmTNF? preferentially activates TNFR2 in monocytic cells and promotes their M2-like differentiation and suppressive activity. Our preliminary data suggest that iNKTs may counteract this process via selective recognition of M2-polarized macrophages. Moreover, crossing NBL-Tag transgenic mice, which spontaneously develop NB, with iNKT-cell deficient animals resulted in an acceleration of tumor growth and an increase of TAM frequency. However, TAMs eventually evade iNKT-cell control, and the evasion is associated with tumor progression. Therefore, we hypothesize that i) tmTNF?-expressing NB cells initiate tumor-supportive inflammation via activation of M2-like TAMs; ii) iNKTs regulate tumor-induced inflammation and indirectly inhibit tumor growth via killing or reprograming of M2-like TAMs; iii) TAMs neutralize iNKTs and enable tumor escape. The following specific aims will test these hypotheses: 1) to examine and therapeutically explore the mechanism by which tmTNF? in NB cells trigger tumor-supportive inflammation; 2) to examine and therapeutically explore the mechanism of reciprocal inhibition between iNKTs and TAMs in the tumor microenvironment. We will use genetic loss of function and gain of function approaches to study the role of NB-derived TNF? isoforms in the activation and functional differentiation of macrophages using in vitro systems with human cells, a syngeneic NB model in mice, and primary tumor specimens from NB patients. To study reciprocal interactions between iNKT and TAMs we recently characterized transgenic NB models with and without genetic deficiency of iNKTs or all NKTs. Finally, we will test whether therapeutic targeting of TAMs maximizes the anti-tumor potential of iNKT-cell immunotherapy for NB. The results are expected to reveal novel mechanisms that govern the initiation and regulation of inflammation in NB and to help identify cellular and molecular targets for the development of effective immunotherapy for NB and other types of cancer.